The catalytic mechanism of Burkholderia cepacia lipase (BCL), which catalyzes the enantioselective hydrolysis of racemic esters of primary alcohols, was investigated by modeling the first stage of the enzymatic hydrolysis of (S/R)-2-methyl-3-phenyl-propanol (MPP) acetate, using molecular dynamics simulations in a mixed quantum mechanical/molecular mechanical (QM/MM) framework. The free energy surface of the enzyme acylation reaction was computed for both enantiomers. The simulations predict the existence of different reaction free energies that favor the (S)-enantiomer over the (R)-enantiomer by 5 kcal/mol. Analysis of the structural and dynamical aspects of the simulated reactions reveals an unforeseen reorganization of the catalytic triad in the (R)-MPP ester, driven by steric hindrance and involving the residues Asp264 and Glu289. Exploiting the different catalytic role of the above-mentioned acidic residues, we suggest a way to regulate the enantioselectivity of BCL by means of a few judicious point mutations that prevent the formation of the second catalytic triad used in the reaction with the (R)-enantiomer. © 2010 American Chemical Society.